The present invention relates to a method for analysis of a sample. More particularly, the present invention relates to a method for three-dimensional analysis of a sample. The present invention also extends to an analyzer for analysis of a sample.
With the advent of scanning probe techniques such as the scanning tunneling microscope STM and the atomic force microscope AFM, it has become possible to image a wide variety of samples with a resolution capability on the molecular or atomic scale.
An AFM image of a sample may be collected by mapping the deflection of a cantilever-mounted probe as a function of the x-y position of the tip of the probe on the sample when the tip comes into contact with, or is in close proximity to, the sample whilst being scanned relative thereto. This yields a topographic image of the sample surface. However, should the sample have an undercut region or steep wall, only the height change and not the profile thereof is accessible by the tip so that the image obtained will not depict the true shape of the sample.
The aforementioned problem has been addressed in an article by Wickramasinghe et. al in Applied Physics Letters, Volume 64(19), published 9 May 1994, pages 2498 to 2500, titled, “Method for imaging sidewalls by atomic force microscopy”. A two-dimensional (2D) AFM is described in this article specifically designed to image samples with vertical profiles. This design involves: (i) the use of a modified tip, specifically, a cylindrical tip fabricated so as to have lower protrusions giving it a “boot” shape; (ii) vibration of the tip in the vertical Z direction and horizontal X direction, (iii) a two-dimensional scan and servo system, and (iv) a mode of acquisition of data points that is different from what is used for conventional AFM imaging.
The method proposed by Wickramasinghe et. al may be applicable to imaging biological samples such as cells, which, not only have vertical edges, typically, for example, at cell boundaries, but also have irregular surfaces. Even so, this method would only yield the possibility of performing a two-dimensional analysis of the profile of such a sample.
Accordingly, it is desirable to provide a method capable of performing a three-dimensional analysis of a sample, particularly a biological sample, which has the atomic/molecular resolution capability of AFM whilst retaining simplicity of design, i.e. not requiring the use of specialized tips, servo- and scanning systems, for example.